1. Field of the Invention
The present invention relates to a method for protein analysis using a water soluble fluorescent compound and the use of the compound as a protein analysis reagent.
2. Background Art
As a method for protein analysis, an electrophoretic methods are widely employed. The electrophoretic methods are advantageous in that these allow proteins to be resolved based on their molecular weights so that the molecular weights can be estimated, these enable semi-quantitative measurement of amounts of proteins, and the methods can be carried out using a relatively simple apparatus, for example.
Examples of a technique for protein analysis using electrophoresis include a technique that involves staining proteins existing in a sample before electrophoresis or directly staining proteins existing on electrophoresis carriers such as gel and then visually detecting the proteins, a technique that involves transferring proteins to an appropriate membrane from electrophoresis carriers and then carrying out protein staining, antibody staining, or the like (e.g., western blotting), and a technique that involves staining proteins existing on electrophoresis carriers, excising a band containing a desired protein and subsequently subjecting the resultant to mass spectroscopy.
Examples of a method for protein staining include a CBB (Coomassie brilliant blue) staining method, a silver staining method, and a Sypro Ruby staining method.
Among these conventional techniques, the CBB method can be carried out conveniently and rapidly, but it has problems that its detection sensitivity is low (detection limit: 50 ng to 100 ng) and its detection sensitivity varies significantly depending on protein type. The silver staining method is a highly sensitive method such that the detection limit ranges from 1 ng to 10 ng, but it has drawbacks in that it cannot be quantitative and requires special processing of a waste solution containing silver. A method using a fluorescent dye such as Sypro Ruby (Invitrogen Corporation) may exhibit higher detection sensitivity than that of the silver staining method; however, when the method is applied to SDS-PAGE, such fluorescent dye tends to be affected by SDS remaining on the carriers, so that a target protein cannot be detected in some cases. Moreover, because of relatively high background, fixation should be carried out before staining proteins with a fluorescent dye as well as washing and decoloration should be carried out for a sufficient period of time after staining. Accordingly, the method has problems that it cannot be performed rapidly and leads to a significant burden on operators.
To solve the above problems with conventional methods for protein staining, the present inventors have searched for novel compounds that can be used for protein staining (International Patent Publication No. WO2006/132030 Pamphlet and JP Patent Publication (Kokai) No. 2007-114009 A). As a result, fluorescent compounds have been obtained that form complexes with proteins via hydrophobic bonds and exhibit a constant rate of color development regardless of protein type (International Patent Publication No. WO2006/132030 Pamphlet). Also, a method for protein detection for electrophoresis has been developed, by which the fluorescent compounds are dissolved in electrophoretic buffers for SDS-PAGE, so as to allow simultaneous performance of protein resolving and protein staining (JP Patent Publication (Kokai) No. 2007-114009 A).
However, all of the fluorescent compounds specifically described in the Examples of the above documents are hardly soluble in water, so that insoluble matter is frequently formed during staining and storage. Therefore, insoluble matter is deposited on electrophoresis carriers and then false positive spots appear during the detection step, which may disturb the detection.